Oral streptococci initiate the formation of the most complex human biofilms, dental plaque; they have also been identified as important pathogens for bacterial endocarditis. Development of dental plaque and vegetation is a biofilm formation process, the ability to form biofilm is critical for bacterial virulence. We have identified a serine-rich glycoprotein Fapl from an oral bacterium, Streptococcus parasanguis and demonstrated that Fapl is required for biofilm formation and bacterial colonization in vivo. Glycosylation of Fapl is important for development of mature biofilm. Fapl-like serine-rich glycoproteins are also found in many pathogenic streptococci and staphylococci and critical for bacterial virulence. To date, little is known about biogenesis of serine-rich glycoproteins. We and others have identified a gene cluster required for biogenesis of serine-rich proteins. Our studies have demonstrated that S. parasanguis modulates its ability to form biofilm by utilizing a specialized Fapl glycosylation system. We hypothesize that Fapl glycosylation and biogenesis is a two-step process. The initial glycosylation step is controlled by a two-component enzyme complex; the subsequent glycosylation step depends on the interaction between a glycosyltransferase and an accessory Sec component. In specific aim #1, we will test the hypothesis that an accessory protein functions as a molecular chaperone to modulate the activity of a glycosyltransferase in the two-component system that initiates the production of a stable functional adhesin. In specific aim #2, we will determine interactions between glycosyltransferase and an accessory Sec component to test our hypothesis that glycosylation and secretion is coupled in the second step. In addition, we will identify protein-protein interaction domains that are important for Fapl glycosylation and bacterial biofilm formation and colonization. We expect completion of this work will lead to a better understanding of Fapl glycosylation and biogenesis and their biological function in bacterial biofilm formation and colonization. As serine-rich proteins are conserved in many streptococcal and staphylococcal pathogens, characterization of Fapl biosynthetic pathway will provide insights on a common theme in glycosylation and biogenesis of serine-rich proteins. This new information on interaction domains required for biofilm formation may lead to the design of therapeutic agent that can disrupt the action of serine-rich glycoproteins and suggest new approaches to prevent and treat bacterial infection. [unreadable] [unreadable] [unreadable]